Proteins are essential components of cells, tissue, and organisms. These macromolecules are made of long strings of amino acids arranged specifically into three dimensional configurations. The side chains of these 22 amino acids create pockets of potential for chemical interactions as the polypeptides fold into their tertiary structures and interact with each other. Proteins initiate and mediate the thousands of biochemical pathways that govern an organism’s function. The careful study of proteins can reveal information about the function of our bodies, the pathways of disease, and the expression of the genetic code. The main challenge to overcome when studying proteins is to choose the most appropriate method of protein extraction.
The main challenge to overcome when studying proteins is to choose the most appropriate method of protein extraction. What tissue are you trying to isolate protein from? Are you interested in studying cytosolic, extracellular, or transmembrane proteins? What about association with a particular subcellular compartment? Do you want to conserve native protein-protein interactions and tertiary/quanternary structure throughout your protein extraction? Do you know specifically which protein you want to isolate, or are you looking for a broad sample? Once you have identified your sample you can tailor your protein extraction to match your experimental goal.
Protein extraction can begin with mechanical or chemical techniques. The initial steps of protein extraction often involve crude mechanical disruption such as cutting, smashing, or shearing tissue into smaller pieces. If intracellular proteins are the target, then detergents can be used to help break apart the phospholipid cellular membrane (cell lysis). Sonication is also used to disrupt the membrane. Often, protease inhibitors are added to the mix to prevent loss of protein due to enzymatic degradation.
Protocols for protein extraction
Extraction is done from tissue or cell culture, tissue requires more steps as there are more layers and heterogeneity across the sample. For tissue, the first step is to mechanically homogenize the sample. Next, centrifugation will give you cytoplasmic proteins. Further you can use chemical buffers to get nuclear proteins. Another chemical buffer containing detergents can be used to obtain membrane proteins. Usually, the cytoskeletal proteins can be extracted last with specific buffers. For cell culture you don’t need to mechanically homogenize your sample but the rest of the procedure is the same, and you begin with lysing your cells. All the buffers used may need to be optimized for different situations and it is important to consider the presence of proteases in your sample. Kits that include buffers for protein extraction are available commercially or are made in laboratories which routinely do protein extraction.
Large pieces of tissue are easily removed by centrifugation after the crude initial tissue disruption. Differential centrifugation often plays an important role in protein extraction; the rate and time of centrifugation will selectively draw subcellular organelles into the pellet. Proteins may thereby be extracted from particular cell compartments via multiple rounds of centrifugation. Additionally, nucleic acids can be chemically precipitated out of the initial tissue slurry and removed by centrifugation. A similar technique can be used to precipitate proteins out of the supernatant. Protein interaction and function are highly dependent on three dimensional structure. Those amino acid side chains provide the chemical pockets that mediate that function via hydrophilic and hydrophobic interactions including hydrogen bonding, ionic, and Van der Waals forces. One way to disrupt these interactions is via a high salt or high/low pH buffer. Once the proteins precipitate out of solution they can be collected by another round of centrifugation. This chemical disruption may not be appropriate for experimental goals that involve investigating native protein-protein interactions as these will be disrupted by the extraction process.
Protein Extraction via affinity
Amino acid side chain chemistry can be exploited to aid protein extraction. An example is an ion-exchange column or a hydrophobic column. There are amino acids with positively charged side chains (arginine, histidine, and lysine), negatively charged side chains (aspartic acid, glutamic acid), and hydrophobic side chains (tyrosine, tryptophan, phenylalanine, valine, isoleucine, etc). These affinities allow proteins to adsorb to a column consisting of a positive, negative, or hydrophobic matrix. Once adsorbed to the column, the proteins can be washed and eluted as an enriched sample.
More specific methods of protein extraction
Protein extraction via centrifugation and affinity columns are effective ways to isolate general proteins in bulk. However, if your experimental goal involves the extraction of a specific protein, then there are other methods that may be more appropriate. This is particularly helpful for antibody or antigen extraction as these proteins have specific affinities for each other. The extraction of IgG antibodies may be performed via protein A/G affinity. Alternatively, specific antibodies can be isolated by attaching their corresponding antigen to a solid surface such as a column or bead. Similarly, the extraction of a specific antigen can be completed by using antibodies containing a recognition site for that antigen. This process is made quick, easy, and efficient with the use of functionalized superparamagnetic nanoparticles and biomagnetic separation.